Reversibly blocked nucleoside analogues and their use

Abstract

Reversibly blocked nucleoside analogues and methods of using such nucleoside analogues for sequencing of nucleic acids are provided.

What is claimed is: 
     
       1. A method of sequencing comprising:
 i) providing a reaction mixture comprising a template nucleic acid, a primer, a polymerase, and a first nucleoside analogue of Formula VI or a first nucleoside analogue of Formula VII: 
 
       
         
           
           
               
               
           
         
         wherein 
         R 1  is a reversible blocking group selected from the group consisting of cyanoethenyl, allenyl, formaldehyde oximyl, acrylaldehyde oximyl, propionaldehyde oximyl, and cyanoethenaldehyde oximyl; 
         X is selected from the group consisting of O and S; 
         R 2  is a nucleobase; 
         L is a cleavable linker; and 
         A 1  of Formula VI comprises an affinity tag; 
         D of Formula VII comprises a detectable fluorescent label; 
         ii) extending the primer by incorporating the first nucleoside analogue of Formula VI with the polymerase; 
         iii) contacting the incorporated first nucleoside analogue with a detectably labeled affinity agent that forms a specific and non-covalent complex with A 1  of the incorporated first nucleoside analogue, thereby specifically labeling the incorporated first nucleoside analogue; and 
         iv) detecting the specifically labeled incorporated first nucleoside analogue of Formula VI; or alternatively, 
         ii) extending the primer by incorporating the first nucleoside analogue of Formula VII with the polymerase; 
         iii) contacting the incorporated first nucleoside analogue with a detectably labeled affinity agent that forms a specific and non-covalent complex with D of the incorporated first nucleoside analogue, thereby specifically labeling the incorporated first nucleoside analogue; and 
         iv) detecting the labeled incorporated first nucleoside analogue by detecting a fluorescence emission from the fluorescent label of Formula VII. 
       
     
     
       2. The method of  claim 1 , wherein the detectably labeled affinity agent is fluorescently labeled, and the detection comprises detecting a fluorescence emission from the fluorescently labeled affinity agent in complex with A 1  of the incorporated first nucleoside analogue. 
     
     
       3. The method of  claim 1 , wherein the method further comprises:
 v) cleaving the reversible blocking group R 1  of the incorporated first nucleoside analogue thereby removing the blocking group from the incorporated first nucleoside analogue; 
 vi) cleaving the linker L thereby removing the affinity tag A 1  of the incorporated first nucleoside analogue, or quenching the label of the detectably labeled affinity agent in complex with A 1  of the incorporated first nucleoside analogue; 
 vii) providing a second nucleoside analogue of Formula VI and a polymerase; 
 viii) extending the primer by incorporating the second nucleoside analogue with the polymerase; 
 ix) contacting the incorporated second nucleoside analogue with a detectably labeled affinity agent that forms a specific and non-covalent complex with A 1  of the incorporated second nucleoside analogue, thereby specifically labeling the incorporated second nucleoside analogue; and 
 x) detecting the specifically labeled incorporated second nucleoside analogue. 
 
     
     
       4. The method of  claim 1 , wherein after detecting the labeled incorporated first nucleoside analogue by detecting a fluorescence emission from the fluorescent label of Formula VII of step (iv), the method further comprising:
 v) cleaving the reversible blocking group R 1  of the incorporated first nucleoside analogue thereby removing the blocking group from the incorporated first nucleoside analogue of Formula VII; 
 vi) providing a second detectably labeled nucleoside analogue of Formula VII and a polymerase; 
 vii) extending the primer by incorporating the second nucleoside analogue of Formula VII with the polymerase; and 
 viii) detecting incorporation of the second nucleoside analogue of Formula VII by detecting a fluorescence emission from the labeled incorporated second nucleoside analogue. 
 
     
     
       5. A method of sequencing comprising:
 i) providing a reaction mixture comprising template nucleic acid, a primer, a polymerase, and a first nucleoside analogue of Formula VII: 
 
       
         
           
           
               
               
           
         
         wherein 
         R 1  is a reversible blocking group selected from the group consisting of cyanoethenyl, allenyl, formaldehyde oximyl, acrylaldehyde oximyl, propionaldehyde oximyl, and cyanoethenaldehyde oximyl; 
         X is selected from the group consisting of O and S; and 
         R 2  comprises a nucleobase; 
         L is a linker; and 
         D comprises a detectable fluorescent label; 
         ii) extending the primer by incorporating the first nucleoside analogue with the polymerase; and 
         iii) detecting the labeled incorporated first nucleoside analogue by detecting a fluorescence emission from the fluorescent label. 
       
     
     
       6. The method of  claim 5 , wherein the method further comprises:
 iv) cleaving the reversible blocking group R 1  of the incorporated first nucleoside analogue thereby removing the blocking group from the incorporated first nucleoside analogue; 
 v) providing a second detectably labeled nucleoside analogue of Formula VII and a polymerase; 
 vi) extending the primer by incorporating the second nucleoside analogue with the polymerase; and 
 vii) detecting incorporation of the second nucleoside analogue by detecting a fluorescence emission from the labeled incorporated second nucleoside analogue.